Method of treating coccidiosis



3,231,467 METHOD OF TREATING COCCHDIOSIS John Martin Clegg, UpperMontciair, Ni, assignor to S. B. Penick & Company, New York, N.Y., acorporation of Deiaware No Drawing. Filed Aug. 1, 1962, Ser. No. 213,8576 Claims. (Cl. 167-531) This invention relates to treatments for thecontrol and mitigation of coccidiosis, a disease which infects poultry,and more particularly it is concerned with dichlorobenzarnides ascoccidiostats and compositions containing such dichlorobenzamides forprophylactic and therapeutic purposes.

Coccidiosis is a widely prevalent problem in the raising of poultry. Itis a protozoan infection attacking the digestive tract and isparticularly toxic to young poultry between the ages of 2 to 14 weeks.The disease is characterized by intestinal disorder, hemorrhage, cecalcongestion, etc., which causes extensive destruction of poultry flocksand substantial economic losses.

It is caused by several species of protozoan parasites of the genus,Eimeria, such as E. tenella, the E. necatrix, E. acervulina, E. maxima,E. hagani, and E. brunetti. E. teneila is the causative agent of acutececal coccidiosis which is a severe and often fatal infection of thececa of the chickens. Manifestations of acute cecal coccidiosis aresevere and extensive hemorrhage, accumulation of blood in the ceca andpassage of blood in the droppings. In untreated birds the mortality mayrun as high as 95%. In addition, survivors of infections arecharacterized by poor Weight gain, poor conformation and reducedfeedefficiency.

E. necatrix and the other species of ooccidia primarily cause theintestinal type of coccidiosis. This may be of either the acute orchronic type; the former often causing death from 5 to 7 days afterinfection and later resulting in a lingering illness.

Chicks heavily infected with cecal or intestinal coccidiosis becomedroopy, look unthrifty, usually have rufiied feathers and pale beaks andshanks. They eventually become so emaciated that death follows. Themortality in a flock is high and may occur very suddenly. A farmersentire investment may be lost in a matter of 2 to 3 weeks. Older birdsthat have become immune to the disease are a constant source ofreinfection as is a previously infested range.

This invention is based on the discovery that dichlorobenzamides havingthe formula:

line

where Cl and Cl may be on any of the positions on the benzene ring; andR is hydrogen or lower alkyl alkenyl, and alkynyl; when administered bythe oral route admixed with the normal nut-riments are effective in thecontrol of coccidiosis in poultry. The compositions consisting of thesedichlorobenzamides admixed in the normal poultry ration have been foundeffective in the treatment of established infections of coccidiosis aswell as in the prevention of such infections.

These coccidiostatic compounds are conveniently fed to poultry as acomponent of the starter mash and in the solid feedstuffs given to themore mature birds. They may also be administered dissolved or suspendedin the drinking water. It is generally convenient and preferred itedStates Pater C the total feed mixture.

3,23 1,46 7 Patented Jan. 25, 1966 "ice to add these dichlorobenzamidesto the poultry feed so that a therapeutic dosage is ingested with thedaily food ration.

The benzarnides may be added directly to the feed as such or in the formof a premix or concentrate. A premix or concentrate of the therapeuticagent in a carrier is commonly employed to facilitate a uniformdispersion of the therapeutic agent in the feed. Suitable carriers forthe premix or concentrate are liquids, such as water, oils, fats, orsolids such as various meals, for example, soybean meal, cornmeal,cottonseed oil meal, sunflower seed meal, linseed oil meal, corncobmeal, or mineral grit, ground oyster shells, all such as commonlyemployed in poultry nutriments. A particularly effective premix carrieris a small portion of the poultry feed itself. In addition to theabovementioned solids, the dichlorobenzamides may be dispersed ordissolved in oily vehicles such as soybean oil, corn oil, cottonseed oiland the like, which are normally added to reinforce poultry mixes andthis material is then blended with the poultry ration.

The high potency concentrates are blended by the feed manufacturer withother feed components in the usual manner. The mixtures are thoroughlyblended and the finished feed should contain up to of grains, up to 30of proteins, and 2 to 4% of minerals. Supplemental vitamins, growthfactors, and antibiotics may also be added as is the current practice.

The proportions of active material in the concentrate are capable ofwide variations since the amount in the finished feed may be adjusted byblending appropriate portions of the premix with the feed to obtain adesired level of therapeutic agent.

For the prevention and treatment of coccidiosis the therapeuticcompounds, i.e., the dichlorobenzamides are employed at low levels infeeds. Depending upon the conditions of exposure, prevalence of thedisease and severity of the epidemic; various proportions of the activecompounds are incorporated into the nutritionally balanced feed asdescribed above. Feed compositions containing as little as 0.005% byweight of the more active dichlorobenzamides will provide prophylacticcontrol for light infections. Concentrations as high as 0.1% by weightmay be employed for short-term intensive therapy. In general it ispreferred to feed the active compound at concentration ranging from 0.01to 0.05% by Weight of Representative feed mixture formulae suitable forthese purposes are to be found in the Feed Trade Manual, published bythe National Provisioner Incorporated; pages 65 through 120 ofthe 1961edition contain a collection of poultry feed formulae.

It has been found that the feeding of the preferred compounds in poultrymixes at the concentrations where the animal ingests from milligrams to500 milligrams of these dichlorobenzamides per kilo of diet will assurefreedom from coccidiosis-induced mortality or debilitation. Thesecompounds at these levels control coccidial infections caused by E.tenella as well as those caused by the other Eimeria species. Thedichlorobenzamides of this invention may be prepared according toprocedures set forth in the representative examples appended below.

Example I .3,5-dichl0robenzamide Prepare 3,5-dichlorobenzoyl chloride byreacting 3,5- dichlorobenzoic acid with an excess of thionyl chloride.Remove the thionyl chloride and purify the acid chloride bydistillation. React the 3,5-dichlorobenzoyl chloride with an excess ofammonia (aqueous) to yield a crude 3,5- dichlorobenzamide. Purify byrecrystallization, M.P. 161-1615 centigrade [see also Ben, 90, pages19-28 (1957)].

ly centigrade.

Example 2.2,3-dicl1lorobenzamide Mix 2,3-dichloroaniline with an excessof hydrochloric acid and sufiicient ice to cool the mixture toapproximate- To this suspension slowly add with stirring sodium nitritedissolved in water. Keep the reaction mixture cool (below 10ccntigrade). Continue the addition of the sodium nitrite until themixture shows a permanent reaction for free nitrous acid (starch-iodidetest paper). Neutralize the acidity of the mixture to a litmus end-pointwith dry sodium carbonate.

Prepare a fresh solution of copper cyanide by reacting copper chloridewith sodium cyanide. Cool this solution to about 0 Centigrade and coverthe surface with a layer of benzene. To this solution slowly add theneutralized diazonium salt. Stir vigorously and keep the reactionmixture cold. Upon completion of the addition, allow the reaction tocontinue in the cold for an additional 30 minutes and then allow themixture to warm to ambient temperatures. centigrade without stirring (toremove dissolved gases). Allow to cool. Separate the two layers. Reservethe benzene layer and extract the aqueous residue with two portions ofbenzene. Steam distill the aqueous layer and extract the distillate withbenzene. Combine the benzene fractions, dry, and distill 0E the benzene.Rccrystallize the residue from petroleum ether (6090) and collect theneedles of 2,3-dichlorobenzonitrile (M.P. 54-56 centigrade). Thiscompound is novel.

Hydrolyze the 2,3-dichlorobenzonitrile with potassium hydroxide in thepresence of methanol and water by refluxing for a few hours. Cool thereaction mixture and harvest the crystals of 2,3-dichlorobenzamide.Recrystallize. The melting point is 16l164 centigrade. This compound isnovel.

Example 3.-2,6-diclzl0r0benzamide Use the procedure of Example 2 aboveby starting with 2,6-dichlorobenzonitrile to prepare2,6-dichlorobenzamide. This material melts at 200-202 centigrade, Reich,Bull. Soc. Chim. (4) 21, 223, in Beilstein, vol. 9, First Supp., page141.

Example 4.-2,4-dichlor0benzamide Use the procedure of Example 1 (secondparagraph) but start with 2,4-dichlorobenzoyl chloride to prepare the2,4-dichlorobenzamide. Melting point l8l-182 centigrade. [A. D. Kirsanovand R. T. Makitra, Zhur Obshchei Khim. 26, page 907-914 (1956).]

Example 5 .2,5 -dichlorobenzamide Use the procedure of Example v1 secondparagraph but start with 2,5-dichlorobenzoyl chloride to prepare the2,5-dichlorobenzamide. This material when recrystallized from water hasa melting point of 155 centigrade (Beilstein, Ann., 179, 290; inBeilstein, vol. 9, 343).

Example 6.-3,4-dichlorobenzamide Use the procedure of Example 1 secondparagraph but start with 3,4-dichlorobenzoyl chloride to prepare the3,4-dichlorobenzamide. Melting point 13-3 centigrade. (Khuhlberg, Ann.,152, 288; in Beilstein, vol. 9, page 344).

Example 7.-3,4-dichloro-N-methylbcnzamide Add3,4-dichloro-N-methylbenzamide in small portions with continuousagitation to an excess of a 40% methylamine solution. Allow the Whitesolid which separates to precipitate. Filter the precipitate, Wash withcold water and then dry it. This 3,4-dichloro-N-methylbenzarnide isrecrystallized from ethyl acetate and melts at 133134 Centigrade. [1.Am. Chem. Soc., 81, 3728 (1959).]

Example 8.-2,S-dichloro-N-methylbenzamide Use the procedure of Example 7but substitute 2,5-

Then slowly heat the mixture to 50 dichlorobenzoyl chloride for the3,4-dichlorobenzoyl chloride of the example. The 2,5-dichloro-N-methylbenzamide melts at l15ll6.5 centigrade.

Example 9.2,5-dichloro-N-ethylbenzamide Use the procedure according toExamples 7 and 8 but substitute ethylamine for the methylamine solutionin Example 7. The 2,S-dichloro-N-ethylbenzamide melts at 89.599.5Centigrade.

Example 10.3,4-dichloro-N-ethylbenzamide Use the procedure of Example 7but substitute ethylamine solution for the methylamine solution ofExample 7. The 3,4-dichloro-N-ethylbenzamide melts at 98-995 centigrade.The lower N-alkyldichlorobenzamides may be prepared in a similar mannerusing the appropriate alkylamine and substituted benzoyl chloride.

The anticoccidial activity is pronounced in all the dichlorobenzamidesincluding those bearing one lower-alkyl, -alkenyl or -alkynylsubstituents having five carbon atoms or less on the amide nitrogen. Theintensity of the anticoccidial effect varies and in the case of the2,4-dichlorobenzamide and 3,5-dichlorobenzamide it is lower than in thecase of the other dichlorobenza-mide isomers. These two isomers, the2,4-dichlorobenzamide and the 3,5-dichlorobenzamide, appear to bedistasteful to chickens and repel them from their feed. Because ofrepulsion, the feed intake is low and the anticoccidial intake of thesecompounds is minimal. Such repulsion can be overcome by masking agentsand/0r attractants.

Specific compounds within the scope of this invention include:

2,3-dichlorobenzamide 2,4-dichlorobenzamide 2,5-dichlorobenzamide2,6-dichlorobenzamide 3,4-dichlorobenzamide 3,5-dichlorobenzamide aswell as the respective:

Dichloro-N-methylbenzamides Dichloro-N-ethylbenzamidesDichloro-N-propylbenzamides Dichloro-N-butylbenzamides andDichloro-N-allylbenzamides Dichloro-N-propargylbenzamides and complexesthereof.

The dichlorobenzamide moiety of this invention appears to be highlyspecific. In contrast to the dichlorobenzamides, themonochlorobenzamides and the trichlorobenzamides possess virtually noanticoccidial activity. Further, extending the carbon chain of theN-alkyl substituents beyond five carbons also appears to eliminate theanticoccidial activity. Other postulated anticoccidial substituents onthe benzene ring such as nitro, in addition to the two chlorine atoms,appear to interfere with the anticoccidial effect. For example,3-nitro-2,5-dichlorobenzamide as well as3-nitro-2,S-dichlormN-ethylbenzamide had no protective etfect againstcoccidiosis infection in chicks, whereas the 2,5-dichlorobenzamide wasone of the more eflective compounds.

Similarly, the 2,4-dichloro-3,5-dinitrobenzamide did not protect thechickens from coccidiosie, although 3,5-dinitrobenzamide hasanticoccidial activity according to US. Patent 3,015,606, issued onJanuary 2, 1962.

In general, the concentration of the active compounds in the medicatedfeed rations should be more than 100 parts per million of ration.Depending on the age of the chicks to which it is administered, andtheir body weight, this corresponds to a daily drug intake of from 5milligrams/kilogram to as much as milligrams/kilogram of body weight.This is based on the assumption that the average chick will ingest up toan equivalent of about 15% of its body weight as its daily food ration.As the chick matures into a chicken the ratio of food intake tobodyweight decreases and the level of 5 milligrams/kilogram of bodyweight of drug is achieved. Of course, the medication may beadministered at higher levels of medication. Such higher concentrations(up to as much as 1% by Weight of feed) may be administered as a singledaily ration if there is evidence of widespread infection in the poultryflock. Such a concentration would yield a medication concentration of1.25 to 1.5 grams of medication per kilogram of body weight in the caseof the average chick just transferred from starter mash to regularfeeds. Such chicks are usually about two weeks old.

Occasionally when it is desired to administer the medication in thewater ration, the medication can be dispersed or emulsified through thedrinking water supply for the poultry flock. The medication can beformulated with non-toxic solvents, oils and sufiicient surfactants toassure even dispersion in the water supply and in concentrationssuflicient to provide adequate dosage of the active ingredients.

Representative dichlorobenzamides have been administered to mammalianspecies and particularly to mice in an attempt to ascertain the LD ofsuch compounds. The mice survived dosages ranging as high a 1 gram of2,4-dichlorobenzamide and 2,5-dichlorobenzamide per kilogram. Thisdemonstrates the lack of toxicity of the compounds and their class andindicates that the materials, when administered to chickens, willpresent little problem of toxic tissue-residues when the poultry isready for market.

The compounds of this invention were administered to two week oldCornish-Rock chicks. The results for the preferred compounds arereported in Table 1.

ing and complete lack of control of the parasite.

parative scores based on the observed symptoms of cecal coccidiosis.

The cecal score is a numerical rating based on the physical findings ofthe cecum on post mortem and is 5 as follows:

1.8No lesions and a functional cecum (1.8 grams is the parison withinoculated unmedicated birds.

2.0Cecum not functioning, mild lesions, reduced in length.

4These scores are based on the average weight per cecum on postmortem.The score of 4 is also assigned to all birds that die of cecalcoccidiosis. Similarly it is 15 the maximum for badly infected birds,indicating complete congestion in the cecum.

The blood score is similarly based on a qualitative comparison withinocculat-ed unmedicated birds.

20 0No blood in the pen-droppings.

While the invention has been illustrated by the foregoing examples ofactive materials, it will be apparent that various equivalent changesand modifications may be resorted to in carrying out this inventionwithout departing from the scope and spirit thereof, and it will beunderstood that such equivalent embodiments are all within the purviewof the annexed claims.

I claim: 1. The method of combating coccidiosis in poultry hus- TABLE I(I? O-NHR Position Percent gain Percent Deaths Cecal Score Blood scoreDrug R p.p.m C1 C1 Medi- Con- Medi- Con- Medi- Oon- Medi- Concated trolcated trol cated trol cated trol 2 3 H 250 69 0 30 1. 8 4. 0 l 4 2 5 H250 47 0 1.8 4. 0 2 5 3 4 H 250 54 37 0 70 2. 0 4. 0 O 4 3 4 CH3 250 7045 O 30 l. 8 3. 0 2 5 3 4 CzH5 250 35 36 40 80 2. 7 4+ 2 4 2 6 H 250 3G45 0 30 1. 8 3. 0 l 4 All birds inoculated with 50,000 oocysts of E.tenella three days after beginning (start) of administration of thedrug. Uninoculated, unmedicated control birds had a weight gain of70-80% during the experimental period.

The chicks were maintained on the medicated feed (starter-mash) and theweight gain ascertained after four days. This provides a measure of thepalatability or toxicity of the medication to uninfected birds. Thechicks were orally inoculated on the fourth day with 50,000 s-porulatedE. tenella oocysts. Surviving chicks were weighed, sacrificed, andpathologically examined on the seventh day after inoculation. Dead birdswere weighed and posted on the day of death.

The percentage Weight gains exhibited by the survivors as compared to:unmedicated inoculated controls is one measure of the anticoccidialactivity. (Uninoculated controls exhibit a weight gain of 70%80%). Othermeasures of anticoccidial activity are the percentage of survivors andthe cecal and blood scores. The percentage of survivors afterinoculation is a self-evident measure of anticoccidial effect. The cecaland blood scores are commilligrams to 1000 milligrams/kilograms of bodyweight of the poultry.

2. The method of combating coccidiosis in poultry which comprisesfeeding to poultry, rations supplying an effective amount of a compoundselected from the group consisting of dichlorobenzamide,dichloro-N-lower alkenyl, dichloro-N-lower-alkynyl, anddichloro-N-loweralkyl substituted benzamide, to combat coccidialinfections.

3. The method according to claim 2 wherein the dichlorobenzamide is2,5-dichlorobenza-mide.

7 4. The method according to claim 2 wherein the dichlorobenzarnide is2,6-dichlorobenzamide.

5. The method according to claim 2 wherein the dichlorobenzamide is3,4-dichlorobenzarnide.

6. The method according to claim 2 wherein the dichlorobenzarnide is2,3-dichl-orobenzamide.

References Cited by the Examiner UNITED STATES PATENTS 2,551,891 5/1951Martin 260558 8 OTHER REFERENCES FRANK CACCIAPAGLIA, JR., LEWIS GOTIS,

Examiners.

1. THE METHOD OF COMBATING COCCIDIOSIS IN POULTRY HUSBANDRY WHICHCOMPRISES THE METHOD OF ORALLY ADMINISTERING TO POULTRY, A COMPOUNDSELECTED FROM THE GROUP CONSISTING OF DICHLOROBENZAMIDE,DICHLORO-N-LOWER-ALKYL, DICHLORO-N-LOWER-ALKYNYL, AND DICHLORO-N-LOWERALKENYLSUBSTITUTED BENZAMIDE, SAID COMPOUND BEING ADMINISTERED IN ADAILY AMOUNT WITHIN THE RANGE OF APPROXIMATELY FIVE MILLIGRAMS TO 1000MILLIGRAMS/KILOGRAMS OF BODY WEIGHT OF THE POULTRY.